CN115078592A - Detection method and application of acrylamide monomer - Google Patents
Detection method and application of acrylamide monomer Download PDFInfo
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- CN115078592A CN115078592A CN202210837025.1A CN202210837025A CN115078592A CN 115078592 A CN115078592 A CN 115078592A CN 202210837025 A CN202210837025 A CN 202210837025A CN 115078592 A CN115078592 A CN 115078592A
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- 238000001514 detection method Methods 0.000 title claims abstract description 53
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 title claims abstract description 31
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims abstract description 82
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 40
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000000178 monomer Substances 0.000 claims abstract description 32
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000003729 cation exchange resin Substances 0.000 claims abstract description 20
- 235000019253 formic acid Nutrition 0.000 claims abstract description 20
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 15
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 14
- 239000001257 hydrogen Substances 0.000 claims abstract description 14
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- 238000004458 analytical method Methods 0.000 claims abstract description 6
- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 claims description 31
- 229960001025 iohexol Drugs 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 28
- 230000014759 maintenance of location Effects 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- 230000000717 retained effect Effects 0.000 claims description 8
- 229920001577 copolymer Polymers 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
- 239000011630 iodine Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 abstract description 11
- 230000010102 embolization Effects 0.000 abstract description 6
- 230000002792 vascular Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 41
- 239000007788 liquid Substances 0.000 description 26
- 239000012488 sample solution Substances 0.000 description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 239000012086 standard solution Substances 0.000 description 15
- 238000011084 recovery Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000003073 embolic effect Effects 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 150000001408 amides Chemical class 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000009969 flowable effect Effects 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical group [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
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- 239000002245 particle Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
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- 150000001336 alkenes Chemical class 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- MYRTYDVEIRVNKP-UHFFFAOYSA-N divinylbenzene Substances C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 125000000542 sulfonic acid group Chemical group 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0031—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/06—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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Abstract
The invention belongs to the technical field of vascular embolization, and particularly relates to a detection method of an acrylamide monomer and application thereof. The detection method provided by the invention comprises the following steps: providing a sample to be detected, wherein the sample to be detected comprises N-isopropylacrylamide and N, N' -methylenebisacrylamide; and (3) carrying out high performance liquid chromatography analysis on a sample to be detected, wherein the chromatographic column is a hydrogen type cation exchange resin chromatographic column, and the mobile phase is a formic acid aqueous solution. The method effectively detects the trace acrylamide monomer remained in the sample: the N-isopropyl acrylamide and the N, N' -methylene bisacrylamide can realize baseline separation between the two trace acrylamide monomers and other components of the sample, and avoid the interference of other components in the sample on the detection background of the trace acrylamide monomers. The method is simple and easy to operate.
Description
Technical Field
The invention belongs to the technical field of vascular embolism, and particularly relates to a detection method of an acrylamide monomer and application of the acrylamide monomer in preparation of poly-N-isopropylacrylamide temperature-sensitive nanogel.
Background
The poly-N-isopropyl acrylamide temperature-sensitive nano gel has lower viscosity in a sol state, is converted into a non-flowable gel state from a good flowing state in a human body temperature environment, has good fluidity and embolization, can overcome the contradiction between the fluidity and the embolization of the traditional embolization agent, has drug-carrying slow-release performance, and is expected to become a new generation of injectable interventional embolization materials.
The poly N-isopropyl acrylamide temperature-sensitive nano gel is a chemical composition, and is prepared by taking N-isopropyl acrylamide as a main monomer and carrying out cross-linking polymerization with other alkene monomers and cross-linking agents such as N, N' -methylene bisacrylamide and the like under the catalytic initiation action of an initiator. Because both N-isopropylacrylamide and N, N '-methylene bisacrylamide have certain biotoxicity, the content of the residual N-isopropylacrylamide and the content of the residual N, N' -methylene bisacrylamide in the product need to be strictly controlled in the research and development and production processes of the poly N-isopropylacrylamide temperature-sensitive nano-gel. The components of the temperature-sensitive nanogel products are complex, and how to detect residual trace N-isopropylacrylamide and N, N' -methylenebisacrylamide in the complex components becomes a technical problem to be solved by technical personnel in the field.
Disclosure of Invention
In view of this, the invention aims to provide a detection method for acrylamide monomers, which ensures effective detection of trace monomers and is applied to research, development and production of poly-N-isopropylacrylamide temperature-sensitive nanogels.
The technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a method for detecting an acrylamide monomer, comprising the following steps:
providing a sample to be tested, wherein the sample to be tested comprises N-isopropyl acrylamide and N, N' -methylene bisacrylamide;
and carrying out high performance liquid chromatography analysis on the sample to be detected, wherein a chromatographic column is a hydrogen type cation exchange resin chromatographic column, and a mobile phase is a formic acid aqueous solution.
According to the method provided by the invention, by adopting the high performance liquid chromatography, utilizing the hydrogen type cation exchange resin chromatographic column and adjusting the mobile phase to be formic acid aqueous solution, the trace acrylamide monomer remained in the sample is effectively detected: n-isopropyl acrylamide and N, N' -methylene bisacrylamide, and enables baseline separation between the two trace acrylamide monomers and other components of the sample, thereby avoiding the interference of other components in the sample on the detection background of the trace acrylamide monomers. The method is simple and easy to operate.
In one embodiment, in the above detection method, the filler of the hydrogen cation exchange resin chromatographic column comprises a hydrogen sulfonated crosslinked styrene-divinylbenzene copolymer.
In one embodiment, in the above detection method, the concentration of formic acid in the aqueous formic acid solution is 0.1% to 5% by mass.
As an embodiment, in the above detection method, the flow rate of the mobile phase is set to 0.4 to 1.2 ml/min.
In one embodiment, in the above detection method, the sample to be detected further includes: iohexol.
Further, the content of iohexol in each milliliter of the sample to be detected is in milligram level calculated by iodine; and/or
The content of the N-isopropylacrylamide and the content of the N, N' -methylenebisacrylamide in each milliliter of the sample to be detected are both in nanogram grade.
Further, the concentration of iohexol is 1-700mgI/ml, the concentration of N-isopropylacrylamide is greater than 7ng/ml, and the concentration of N, N' -methylenebisacrylamide is greater than 7 ng/ml.
As an embodiment, in the above detection method, the step of subjecting the sample to be detected to high performance liquid chromatography further includes: and injecting the mobile phase after passing through the hydrogen type cation exchange resin chromatographic column into a mass spectrometer for mass spectrometry.
Further, a mobile phase after passing through the hydrogen-type cation exchange resin chromatographic column is retained according to retention times of the iohexol, the N-isopropylacrylamide and the N, N '-methylenebisacrylamide, and a retained solution corresponding to the N-isopropylacrylamide and the N, N' -methylenebisacrylamide is obtained.
In a second aspect, the invention also provides application of the detection method in preparation of the poly-N-isopropylacrylamide temperature-sensitive nanogel.
The detection method of the acrylamide monomer can effectively detect the trace acrylamide monomer remained in the sample: the N-isopropylacrylamide and the N, N '-methylenebisacrylamide have high recovery rate, the relative standard deviation is less than 15 percent, the requirement on the accuracy of trace substance detection is met, and the N-isopropylacrylamide and the N, N' -methylenebisacrylamide are applied to the preparation of the poly-N-isopropylacrylamide temperature-sensitive nanogel, so that the content of the residual acrylamide monomer in the product can be controlled, and the biological safety of the product can be ensured.
Drawings
FIG. 1 is a liquid chromatogram of a sample solution to be tested in example 1;
FIG. 2 is a liquid chromatogram of a MBAM standard solution in example 1;
FIG. 3 is a liquid chromatogram of a NIPAM standard solution of example 1;
FIG. 4 is a liquid chromatogram of a sample solution to be tested in example 2;
FIG. 5 is a liquid chromatogram of a sample solution to be tested in example 3;
FIG. 6 is a liquid chromatogram of a sample solution to be tested in example 4;
FIG. 7 is a liquid chromatogram of a sample solution to be tested in example 5;
FIG. 8 is a liquid chromatogram of the sample solution to be measured in comparative example 1;
fig. 9 shows the ratio of acetonitrile: a liquid chromatogram recorded by taking a formic acid solution (5:95) with pH2.5 as a mobile phase;
fig. 10 shows the sample solution to be tested in comparative example 2 in acetonitrile: the pH2.5 formic acid solution (10:90) is the liquid chromatogram recorded for the mobile phase.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides a method for detecting an acrylamide monomer, which comprises the following steps:
s01, providing a sample to be detected, wherein the sample to be detected comprises N-isopropyl acrylamide and N, N' -methylene bisacrylamide;
and S02, carrying out high performance liquid chromatography analysis on the sample to be detected, wherein the chromatographic column is a hydrogen type cation exchange resin chromatographic column, and the mobile phase is formic acid aqueous solution.
According to the method provided by the embodiment of the invention, by adopting the high performance liquid chromatography, utilizing the hydrogen type cation exchange resin chromatographic column and adjusting the mobile phase to be formic acid aqueous solution, the trace acrylamide monomer remained in the sample is effectively detected: n-isopropyl acrylamide and N, N' -methylene bisacrylamide, and enables baseline separation between the two trace acrylamide monomers and other components of the sample, thereby avoiding the interference of other components in the sample on the detection background of the trace acrylamide monomers. The method is simple and easy to operate.
Specifically, in step S01, the sample to be tested contains N-isopropylacrylamide and N, N' -methylenebisacrylamide. In the synthesis of the poly N-isopropyl acrylamide temperature-sensitive nano gel, N-isopropyl acrylamide is used as a main monomer, and N, N' -methylene bisacrylamide is used as a cross-linking agent, so that the synthesized poly N-isopropyl acrylamide temperature-sensitive nano gel is a cross-linked polymer with a three-dimensional network structure, has more excellent embolization performance compared with linear poly N-isopropyl acrylamide temperature-sensitive nano gel, has lower viscosity in a sol state, and is converted from a good flowing state into a non-flowable gel state in a human body temperature environment.
The sample to be detected in the embodiment of the invention can be derived from poly N-isopropyl acrylamide temperature-sensitive nanogel, and can also be derived from temperature-sensitive nanogel embolic agent formed by compounding poly N-isopropyl acrylamide temperature-sensitive nanogel with other components such as iohexol and the like. It can be understood that the sample to be detected in the embodiment of the present invention has the temperature sensitive nanogel removed, so as to avoid adverse effects of the temperature sensitive nanogel on the liquid chromatography column, such as blocking the chromatography column.
In some embodiments, the sample to be tested further comprises: iohexol. The iohexol is a developer in the temperature-sensitive nano-gel embolic agent, has extremely high concentration relative to trace acrylamide monomers remained in a sample, has great interference on the detection background of the trace acrylamide monomers due to the existence of the iohexol, and is difficult to effectively detect the N-isopropylacrylamide and the N, N' -methylenebisacrylamide remained in the temperature-sensitive nano-gel embolic agent by adopting the conventional high performance liquid chromatography method. The embodiment of the invention effectively overcomes the technical problem by utilizing the hydrogen type cation exchange resin chromatographic column and the method for adjusting the mobile phase to be the formic acid aqueous solution.
In some embodiments, the amount of iohexol per ml of sample to be tested is on the milligram scale as iodine. On the basis, preferably, the content of N-isopropylacrylamide and the content of N, N' -methylenebisacrylamide in each milliliter of the sample to be detected are both in nanogram grade. By the detection method provided by the embodiment of the invention, baseline separation between the trace residual amide components at the nanogram level and the iohexol at the milligram level can be realized.
It is understood that milligram scale means an amount within 1-1000mg and nanogram scale means an amount within 1-1000 ng.
In the specific embodiment, the concentration of iohexol is 1-700mgI/ml, the concentration of N-isopropylacrylamide is greater than 7ng/ml, and the concentration of N, N' -methylenebisacrylamide is greater than 7 ng/ml. The detection limit of the detection method provided by the embodiment of the invention on the monomer can reach 7ng/ml, the requirement of most application scenes on the detection of trace substances is met, and the application range is wide.
Step S02 is an execution step of performing high performance liquid chromatography on the sample to be tested, where the chromatographic column is a hydrogen-type cation exchange resin chromatographic column, and the mobile phase is an aqueous formic acid solution.
In some embodiments, the packing material of the hydrogen cation exchange resin chromatography column comprises a hydrogen sulfonated cross-linked styrene-divinylbenzene copolymer. In a specific embodiment, the hydrogen-type cation exchange resin chromatographic column is a hydrogen-type sulfonated crosslinked styrene-divinylbenzene copolymer cation exchange resin chromatographic column. Such columns are commonly used for separating carbohydrates/sugars and acids, for example for measuring benzoic acid in milk. The applicant creatively applies the chromatographic column to the analysis and detection of acrylamide monomers in poly-N-isopropylacrylamide temperature-sensitive nano-gel, realizes the detection of trace residual amide components at microgram/nanogram level by optimizing the condition of a mobile phase, and realizes the baseline separation of the trace residual amide components and iohexol at milligram level. In addition, the filler is granular, and mainly comprises polystyrene/divinylbenzene copolymer particles modified with active hydrogen-containing groups (such as sulfonic acid groups) on the surface. Further, the degree of crosslinking of the filler is about 3% to 10%, e.g., 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; the copolymer particles are 1-10 μm, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μm.
In some embodiments, the aqueous formic acid solution has a formic acid concentration of 0.1% to 5% by weight. In particular embodiments, the concentration is 0.1%, 0.13%, 0.15%, 0.18%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5%. Among them, the pH of the aqueous formic acid solution is preferably 1 to 3.
In some embodiments, the flow rate of the mobile phase is set to 0.4-1.2 ml/min. In particular embodiments, the flow rate is 0.4ml/min, 0.5ml/min, 0.6ml/min, 0.7ml/min, 0.8ml/min, 0.9ml/min, 1.0ml/min, 1.1ml/min, or 1.2 ml/min.
In some embodiments, after the step of subjecting the sample to be tested to high performance liquid chromatography, the method further comprises: injecting the mobile phase after passing through the hydrogen type cation exchange resin chromatographic column into a mass spectrometer for mass spectrum analysis. By combining the mass spectrometry, on one hand, the monomer separated by the chromatographic column can be qualitatively detected, and the target monomer is secondarily confirmed: n-isopropylacrylamide and N, N' -methylenebisacrylamide; on the other hand, other data given by mass spectrum can be used for measuring the content of the residual monomer, so that the problems that the high performance liquid chromatography has high detection limit, cannot effectively detect low-concentration components (such as nanogram level) and cannot carry out quantification according to a liquid chromatogram are solved.
In a further example, the mobile phase after passing through a column of cation exchange resin in hydrogen form was retained according to the retention time of iohexol, N-isopropylacrylamide and N, N '-methylenebisacrylamide, to obtain a retained solution corresponding to N-isopropylacrylamide and N, N' -methylenebisacrylamide. In a specific embodiment, an entrapment solution corresponding to N-isopropylacrylamide and an entrapment solution corresponding to N, N' -methylenebisacrylamide are combined, and then a predetermined volume is determined to obtain a monomer entrapment solution. The method is applied to a scene when the concentration of the acrylamide monomer is less than the detection limit of the high performance liquid chromatography, solves the problem that a chromatographic peak cannot be effectively shown in a high performance liquid chromatogram of a low-concentration component (which is lower than the detection limit), effectively utilizes mass spectrum data to carry out quantitative detection, and realizes the detection of nanogram-level residual monomers.
In conclusion, by the detection method, the trace acrylamide monomer remained in the temperature-sensitive nano gel embolic agent can be effectively detected: n-isopropyl acrylamide and N, N' -methylene bisacrylamide, and effectively reduces the interference of other components iohexol on the detection background, and the method is simple and easy to operate. In addition, the detection limit of the detection method on the acrylamide monomer can reach 7ng/ml, the quantification limit can reach 20ng/ml, the recovery rate of N, N' -methylene-bisacrylamide can reach 91.9%, the recovery rate of N-isopropylacrylamide can reach 86.7%, the relative standard deviation of the recovery rates is less than 15%, and the requirement on the accuracy of trace substance detection is met.
On the basis of the technical scheme, the embodiment of the invention also provides application of the detection method in preparation of the poly N-isopropyl acrylamide temperature-sensitive nano gel.
The detection method of the acrylamide monomer can effectively detect the trace acrylamide monomer remained in the sample: the N-isopropylacrylamide and N, N '-methylenebisacrylamide have high recovery rates, the relative standard deviation of the recovery rates is less than 15%, the requirement on the accuracy of trace substance detection is met, and the N-isopropylacrylamide and N, N' -methylenebisacrylamide are applied to the preparation of the poly-N-isopropylacrylamide temperature-sensitive nano-gel, so that the content of residual acrylamide monomers in the product can be controlled, and the biological safety of the product can be ensured.
In order to make the details and operation of the above-mentioned embodiments of the present invention clearly understood by those skilled in the art, and to make the progress of the method for detecting an acrylamide-based monomer and its use obvious, the following examples illustrate the embodiments of the present invention.
In the following examples, N-isopropylacrylamide is denoted NIPAM and N, N' -methylenebisacrylamide is denoted MBAM.
Example 1
S11, preparing a sample to be detected: dissolving iohexol, NIPAM and MBAM in water to form a mixed solution serving as a sample solution to be detected; the concentration of iohexol in the solution was greater than 300mgI/ml, the concentration of NIPAM was 10. mu.g/ml and the concentration of MBAM was 10. mu.g/ml.
Meanwhile, respectively preparing NIPAM standard substance solution (1mg/ml) and MBAM standard substance solution (1 mg/ml);
s12, high performance liquid chromatography
(1) Chromatographic conditions
A chromatographic column: sulfonated crosslinked styrene-divinylbenzene copolymer hydrogen type cation exchange resin chromatographic column,
mobile phase: an aqueous formic acid solution (formic acid content equivalent to 0.19%) at pH2.5,
detection wavelength: 220nm, column temperature: 60 ℃, sample introduction: 100 μ L, flow rate: 1.0 ml/min.
(2) By adopting the chromatographic conditions, the sample solution to be detected, the NIPAM standard solution and the MBAM standard solution are respectively injected into the Shimadzu LC-20A of the liquid chromatograph for analysis and detection to obtain chromatograms shown in figures 1-3, and by combining the results of the liquid chromatograms of the two standard solutions in figures 2-3, the second chromatographic peak in figure 1 can be determined to correspond to MBAM, the third chromatographic peak to correspond to NIPAM, and the first chromatographic peak to correspond to iohexol.
The separation degrees of the two monomers of the MBAM and the NIPAM in the figure 1, the retention time and the signal to noise ratio of the MBAM and the NIPAM are counted to obtain the results in the table 1:
TABLE 1
Example 2
The chromatographic conditions used in this example differ from those of example 1 in that: the flow rate is 0.4ml/min, and the obtained liquid chromatogram of the sample solution to be tested is shown in FIG. 4.
The separation degrees of the two monomers, i.e., MBAM and NIPAM, as well as the retention times and signal-to-noise ratios of MBAM and NIPAM in FIG. 4 were counted to obtain the results shown in Table 2:
TABLE 2
Example 3
The chromatographic conditions used in this example differ from those of example 1 in that: the mobile phase is 0.1% formic acid water solution, and the liquid chromatogram of the obtained sample solution to be tested is shown in fig. 5.
The separation degrees of the two monomers MBAM and NIPAM in FIG. 5, the retention times and the signal-to-noise ratios of MBAM and NIPAM were counted to obtain the results in Table 3:
TABLE 3
Example 4
The chromatographic conditions used in this example differ from those of example 3 in that: the flow rate is 0.4ml/min, and the obtained liquid chromatogram of the sample solution to be tested is shown in FIG. 6.
The separation degrees of the two monomers MBAM and NIPAM in FIG. 6, the retention times and the signal-to-noise ratios of MBAM and NIPAM were counted to obtain the results in Table 4.
TABLE 4
Example 5
S51, preparing a sample to be detected: dissolving iohexol, NIPAM and MBAM in water to form a mixed solution serving as a sample solution to be detected; the concentration of iohexol in the solution was 300mgI/ml, the concentration of NIPAM was 50ng/ml and the concentration of MBAM was 50 ng/ml.
Meanwhile, NIPAM standard solution (0.5ng/ml) and MBAM standard solution (0.5ng/ml) were prepared separately.
S52, high performance liquid chromatography
The chromatographic conditions were the same as in example 1.
And (3) operating the high performance liquid chromatograph, injecting the sample solution to be detected into the liquid chromatograph, and obtaining the liquid chromatogram in the figure 7, wherein chromatographic peaks of NIPAM and MBAM are not seen in the figure because the concentrations of the NIPAM and the MBAM are lower than the detection limit of the liquid chromatogram.
S53, column passing interception and preparation of monomer interception solution
The pipeline at the inlet of the high performance liquid chromatograph detector is taken down, and the pipeline port is placed in a clean beaker, so that the mobile phase passing through the column does not enter the detector. The sample solution to be tested prepared in example 1 was injected into a liquid chromatograph. Referring to the retention times shown in FIG. 1 of example 1, mobile phases corresponding to N, N' -methylenebisacrylamide and N-isopropylacrylamide were respectively retained, and the retained solutions were collected and placed in the same brown volumetric flask, and allowed to stand at room temperature, and the volume was increased to 10ml with the mobile phase, and were used as a monomer-retaining solution for future use.
S54 detection by liquid phase-mass spectrometry
And (4) respectively injecting the monomer interception solution prepared in the step S53, the NIPAM standard solution prepared in the step S51 and the MBAM standard solution into a mass spectrum for sample injection detection, and counting the recovery rates of the NIPAM and the MBAM, wherein the counting results are shown in Table 5.
The formula:
the measured value of the sample content is 100 x (average value of sample peak area/average value of control peak area) x the concentration of the control,
the sample recovery rate (measured sample content/theoretical sample preparation value) × 100%, and the theoretical sample preparation value is the concentration prepared in step S51.
TABLE 5
Note: the sample group corresponds to the monomer retention solution, and the control group corresponds to the NIPAM standard solution and the MBAM standard solution.
Example 6
S61, preparing a sample to be detected: dissolving iohexol, NIPAM and MBAM in water to form a mixed solution serving as a sample solution to be detected; the concentration of iohexol in the solution was 300mg/ml, the concentration of NIPAM was 10. mu.g/ml and the concentration of MBAM was 10. mu.g/ml.
Meanwhile, NIPAM standard solution (100ng/ml) and MBAM standard solution (100ng/ml) are prepared respectively.
S62 and S63 similar to example 5, the recovery rates of NIPAM and MBAM were counted, and the statistical results are shown in Table 6.
TABLE 6
Note: the sample group corresponds to the monomer retention solution, and the control group corresponds to the NIPAM standard solution and the MBAM standard solution.
Comparative example 1
1. The step of preparing a sample solution to be tested is the same as step S11 of example 1;
2. the following chromatographic conditions were used:
HPLC-UV (Agilent 1260)
A chromatographic column: c18 column
Mobile phase: acetonitrile: water 10:90 detection wavelength: 220nm
Column temperature: sample introduction at 35 ℃: 100 μ L
Flow rate: 1.0ml/min
Operating a high performance liquid chromatograph, taking 100 mu L of sample solution to be detected, injecting the sample solution into the liquid chromatograph, and sequentially taking iohexol (3-4min), N, N '-methylene bisacrylamide (6min) and N-isopropyl acrylamide (10min) as shown in figure 9, wherein the chromatographic peak trailing phenomenon of iohexol is obvious, and baseline separation between the N, N' -methylene bisacrylamide and iohexol is not realized.
Comparative example 2
The chromatographic conditions used in this comparative example differ from those of example 2 in that: the mobile phase was adjusted to acetonitrile: pH2.5 formic acid solution (5:95 and 10: 90).
The results are shown in FIGS. 9-10, where there was no significant change in the time to peak of iohexol, forward shift in the retention time of the two monomers, and no significant optimization of the signal to noise ratio when 5% or 10% acetonitrile was added to the mobile phase. As in comparative example 1, the chromatographic peak tailing phenomenon of iohexol was significant, and no baseline separation was achieved between NIPAM and iohexol.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A detection method of acrylamide monomers comprises the following steps:
providing a sample to be tested, wherein the sample to be tested comprises N-isopropyl acrylamide and N, N' -methylene bisacrylamide;
and carrying out high performance liquid chromatography analysis on the sample to be detected, wherein the chromatographic column is a hydrogen type cation exchange resin chromatographic column, and the mobile phase is a formic acid aqueous solution.
2. The detection method according to claim 1, wherein the filler of the hydrogen-type cation exchange resin chromatographic column comprises a hydrogen-type sulfonated crosslinked styrene-divinylbenzene copolymer.
3. The detection method according to claim 1, wherein the concentration of formic acid in the aqueous formic acid solution is 0.1 to 5% by mass.
4. The detection method according to claim 1, wherein the flow rate of the mobile phase is set to 0.4 to 1.2 ml/min.
5. The method according to any one of claims 1 to 4, wherein the sample to be tested further comprises: iohexol.
6. The detection method according to claim 5, wherein the content of iohexol in each ml of the sample to be detected is on the milligram scale in terms of iodine; and/or
The content of the N-isopropylacrylamide and the content of the N, N' -methylenebisacrylamide in each milliliter of the sample to be detected are both in nanogram grade.
7. The detection method according to claim 6, wherein the concentration of iohexol is 1 to 700mgI/ml, the concentration of N-isopropylacrylamide is greater than 7ng/ml, and the concentration of N, N' -methylenebisacrylamide is greater than 7 ng/ml.
8. The detection method according to claim 6, wherein the step of subjecting the sample to be detected to high performance liquid chromatography further comprises: and injecting the mobile phase after passing through the hydrogen type cation exchange resin chromatographic column into a mass spectrometer for mass spectrometry.
9. The detection method according to claim 8, wherein a mobile phase passing through the hydrogen-type cation exchange resin chromatographic column is retained according to retention times of the iohexol, the N-isopropylacrylamide and the N, N '-methylenebisacrylamide to obtain a retained solution corresponding to the N-isopropylacrylamide and the N, N' -methylenebisacrylamide.
10. The use of the detection method according to any one of claims 1 to 9 in the preparation of poly-N-isopropylacrylamide temperature-sensitive nanogels.
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